Journal: Molecular Cancer

Article Title: Hedgehog signaling drives glial cell plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine neoplasms

doi: 10.1186/s12943-026-02611-y

Figure Lengend Snippet: Patient-derived PanNET tumoroids express HH signaling proteins and respond to HH pathway activation and inhibition. A Combined fluorescence and phase-contrast images of 21-day-old human PanNET tumoroids (PanNET3) stained for chromogranin A (CHGA) and HH proteins (PTCH1, SHH). B Four patient-derived PanNET tumoroid lines (PanNET1–4) were exposed to recombinant human SHH N-terminal peptide (100 ng/mL) or the SMO inhibitor vismodegib (VISMO, 20 µM) for 72 h and changes in mRNA expression were analyzed by RT-qPCR. mRNA changes were normalized to HPRT1 expression and DMSO vehicle control. ( n = 4 unique patient lines). * = p < 0.05, ** = p < 0.01, **** = p < 0.0001 by Two-way ANOVA with Sidak post-test. C EdU labeling showing proliferation of dissociated PanNET3 cells following 5-day exposure to: the HH agonists SHH-N (100 ng/mL) and SAG (10 nM); inhibitors of the canonical HH signaling pathway vismodegib (20 µM) and sonidegib (10 nM); or inhibitors of the GLI1/2 effectors GANT61 (10 µM) and itraconazole (ITZ, 1 µM). D Quantitation of the percentage of EdU-positive PanNET tumor cells following 5-day treatment. ( n = 3 replicates from one patient tumoroid line). * = p < 0.05, by One-way ANOVA with Tukey post-test. E SHH or SAG were co-administered with the respective pharmacologic inhibitors and EdU uptake was evaluated after 7 days. F Immunofluorescent images of CHGA, SHH, and PTCH1 expression in a second PanNET tumoroid line (PanNET5). G , H EdU labeling was evaluated in PanNET5 tumoroids following 7-day treatment with SHH-N (200 ng/mL) and SAG (20 nM); inhibitors of the canonical HH signaling pathway vismodegib (20 µM) and sonidegib (10 nM); or inhibitors of the GLI1/2 effectors GANT61 (10 µM) and itraconazole (ITZ, 5 µM). I Crystal violet staining of human BON-1 PanNET cells after 48 h treatment. J BrdU incorporation in BON-1 cells after 48 h treatment with HH agonists SHH-N and SAG, ( K ) SMO inhibitors vismodegib and sonidegib, and ( L ) inhibitors of GLI1/2 signaling. M Immunofluorescent images of CHGA, SHH, and PTCH1 expression in tumoroids derived from a metastatic ileal NET (IL-NET-met1). N , O EdU labeling was assayed in the IL-NET-met1 tumoroid line following 7-day treatment with the same drug concentrations used for PanNET5. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 by One-way ANOVA with Dunnett post-test

Article Snippet: Drug compounds used in the studies include: human recombinant SHH N-terminal peptide (R&D Systems, Cat# 1845-GMP), SAG (Tocris, Cat# 4366), vismodegib (Tocris, Cat# 7710), sonidegib (Tocris, Cat# 7826), GANT61 (Tocris, Cat# 3191), and itraconazole (Tocris, Cat# 5981).

Techniques: Derivative Assay, Activation Assay, Inhibition, Fluorescence, Staining, Recombinant, Expressing, Quantitative RT-PCR, Control, Labeling, Quantitation Assay, BrdU Incorporation Assay

Journal: Molecular Cancer

Article Title: Hedgehog signaling drives glial cell plasticity and oncogenic reprogramming in gastroenteropancreatic neuroendocrine neoplasms

doi: 10.1186/s12943-026-02611-y

Figure Lengend Snippet: Loss of Men1 in enteric glial cells stimulates GLI1/2-dependent transcriptional reprogramming. A Combined fluorescence and phase contrast images of 5-day-old primary enteric glial cell (EGC) cultures from Sox10-CreER T2 ; LSL-tdTomato mice and CreER T2 negative controls. Top panel shows TdTomato+ EGCs after 48 h exposure to 4-hydroxytamoxifen 4-OHT (2 µM). B TdTomato + EGCs were sorted by FACS to enrich for a pure SOX10 + cell population. C Combined fluorescence and phase contrast images of FACS-enriched SOX10-tdTomato + EGCs. D Fluctuations in HH pathway mRNA levels were evaluated in SOX10-tdTomato + EGCs 72 h following siRNA-mediated Men1 silencing. siRNA treatment consisted of four pooled siRNAs targeting the Men1 gene ( si -Men1 , 25 nM) or non-targeting (si-NT, 25 nM) controls. ( n = 5). E Immunofluorescence images of SHH expression in si-NT and si-Men1 treated EGCs (SHH = red pseudo-color, DAPI = blue). Inset shows higher power image. F Western blot analysis of si-NT and si-Men1 EGCs after 72 h treatment. SHH-FL = 55 kDa full length peptide; SHH- N = 22 kDa N-terminal peptide. ( n = 3). G Quantitation of protein expression in panel (F) normalized to GAPDH loading control. ( n = 3). H Relative fold-change in glial lineage transcripts and ( I ) neuroendocrine and neural progenitor transcripts in si-NT and si-Men1 treated EGCs. ( n = 6). J qPCR analysis of HH pathway genes and ( K ) neuroendocrine and neural progenitor transcriptsin si-NT and si-Men1 EGCs after 72 h treatment with GANT61 (10 µM) or vismodegib (VISMO 20 µM). ( n = 3). For all plots, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 by Two-way ANOVA with Sidak post-test. L Immunofluorescence images of si-NT and si-Men1 EGCs after 96 h siRNA knockdown and 72 h treatment with vehicle or GANT61. Menin = green, GFAP = magenta, SHH = yellow. M Significant GSEA pathways in enteric glial cells following 5-days of si- Men1 knockdown compared to non-targeting control. GSEA was performed on Men1 -depleted cells and cells co-treated with ( N ) si- Men1 , si- Gli1 , and si- Gli2 , and ( O ) cells co-treated with si- Men1 and GANT61 (10 µM). P – R KEGG pathway enrichment analysis comparing the same groups as shown in panels ( M – O ). S Heatmap showing significant DEGs mapped to the cell cycle, HH signaling, epigenetic regulation, neural stem cell (NSC) reprogramming, neuronal and neuroendocrine differentiation

Article Snippet: Drug compounds used in the studies include: human recombinant SHH N-terminal peptide (R&D Systems, Cat# 1845-GMP), SAG (Tocris, Cat# 4366), vismodegib (Tocris, Cat# 7710), sonidegib (Tocris, Cat# 7826), GANT61 (Tocris, Cat# 3191), and itraconazole (Tocris, Cat# 5981).

Techniques: Fluorescence, Immunofluorescence, Expressing, Western Blot, Quantitation Assay, Control, Knockdown